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Abstract

Silencing in the yeast Saccharomyces cerevisiae is known at three classes of loci: at the silent mating-type loci HML and HMR, in subtelomeric regions and at the highly repetitive rDNA locus, which resides in the nucleolus. rDNA silencing differs markedly from the other two classes of silencing in that it requires a DNA-associated protein complex termed RENT. The Net1 protein, a central component of RENT, is required for nucleolar integrity and the control of exit from mitosis. Another RENT component is the NAD+-dependent histone deacetylase Sir2, which is the only silencing factor known to be shared among the three classes of silencing. Here, we investigated the role of Net1 in HMR silencing. The mutation net1-1 as well as NET1 expression from a 2μ-plasmid restored repression at silencing defective HMR loci. Both effects were strictly dependent on the Sir proteins. We found overexpressed Net1 protein to be directly associated with the HMR-E silencer, suggesting that Net1 could interact with silencer binding proteins (for instance, Rap1 and the origin recognition complex, ORC) and recruit other silencing factors to the silencer. Furthermore, we found that ORC may have a role in RENT recruitment to the rDNA. Moreover, Net1 provided ORC dependent , Sir1 independent silencing when artificially tethered to the HMR-E silencer. Conversely, our data suggested that net1-1 acted indirectly in HMR silencing by releasing Sir2 from the nucleolus, thus shifting the internal competition for Sir2 between the silenced loci towards HMR.